Research article 5211 Fgf8 expression defines a morphogenetic center required for olfactory neurogenesis and nasal cavity development in the mouse Shimako Kawauchi1, Jianyong Shou1,*, Rosaysela Santos1, Jean M. Hébert2, Susan K. McConnell3, Ivor Mason4 and Anne L. Calof1,† 1Department of Anatomy and Neurobiology, and Developmental Biology Center, University of California, Irvine, CA 92697-1275, USA 2Department of Neuroscience and Molecular Genetics, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, USA 3Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA 4MRC Centre for Developmental Neurobiology, New Hunt’s House, King’s College London, Guy’s Campus, London SE1 1UL, UK *Present address: Lilly Research Labs-Functional Genomics, Eli Lilly and Company, Indianapolis, IN 46285, USA †Author for correspondence (e-mail: [email protected])

Accepted 23 September 2005

Development 132, 5211-5223 Published by The Company of Biologists 2005 doi:10.1242/dev.02143

Summary In vertebrate olfactory epithelium (OE), neurogenesis domain undergo high levels of apoptosis, resulting in proceeds continuously, suggesting that endogenous signals cessation of nasal cavity invagination and loss of virtually support survival and proliferation of stem and progenitor all OE neuronal cell types. These findings demonstrate that cells. We used a genetic approach to test the hypothesis that Fgf8 is crucial for proper development of the OE, nasal Fgf8 plays such a role in developing OE. In young embryos, cavity and VNO, as well as maintenance of OE Fgf8 RNA is expressed in the rim of the invaginating nasal neurogenesis during prenatal development. The data pit (NP), in a small domain of cells that overlaps partially suggest a model in which Fgf8 expression defines an with that of putative OE neural stem cells later in gestation. anterior morphogenetic center, which is required not only In mutant mice in which the Fgf8 gene is inactivated in for the sustenance and continued production of primary anterior neural structures, FGF-mediated signaling is olfactory (OE and VNO) neural stem and progenitor cells, Development strongly downregulated in both OE proper and underlying but also for proper morphogenesis of the entire nasal cavity. mesenchyme by day 10 of gestation. Mutants survive gestation but die at birth, lacking OE, vomeronasal organ Key words: FGF, Vomeronasal organ, Neurogenesis, Olfactory (VNO), nasal cavity, forebrain, lower jaw, eyelids and epithelium, Nasal cavity, Stem cell, Apoptosis, Cre recombinase, pinnae. Analysis of mutants indicates that although initial Fgf8, Foxg1, Sox2, Pax6, Mash1, Neurogenin 1, Ncam1, Pyst1, Shh, NP formation is grossly normal, cells in the Fgf8-expressing Dlx5, Neuronal progenitor, Mouse mutant

Introduction 2003; Garel et al., 2003; Grove and Fukuchi-Shimogori, 2003; Members of the fibroblast growth factor (FGF) superfamily of Irving and Mason, 2000; Ohkubo et al., 2002; Trainor et al., signaling molecules have important effects on cell 2002). More recently, the idea has emerged that FGF8 is proliferation, developmental patterning, cell growth and involved in survival and/or maintenance of specific developing homeostasis in virtually all tissues in higher vertebrates cell populations that ultimately give rise to particular (Ornitz, 2000). In the nervous system, studies in vitro and in components of the nervous system (Chi et al., 2003; Mathis et vivo have shown that FGFs promote proliferation, al., 2001; Storm et al., 2003). differentiation and survival of most neural cell types, including Mouse olfactory epithelium (OE) provides a useful model stem cells and neuronal progenitors (DeHamer et al., 1994; system with which to understand how neurogenesis is Eckenstein, 1994; Ford-Perriss et al., 2001; Reuss and von regulated at the cellular and molecular levels. Studies in vitro Bohlen und Halbach, 2003; Temple and Qian, 1995). FGF8 in and in vivo have demonstrated four distinct developmental particular appears to play an important role in developing stages in the neuronal lineage of established OE: neural stem nervous system, although the mechanism(s) by which it acts cells, which express the transcription factor Sox2; Mash1 have not been elucidated fully. FGF8 was first identified as a (Ascl1 – Mouse Genome Informatics)-expressing committed mitogen (Tanaka et al., 1992), and some data support a neuronal progenitors, the progeny of the stem cells; Ngn1- mitogenic role for FGF8 in neural tissues (Xu et al., 2000; Lee expressing immediate neuronal precursors (INPs), the progeny et al., 1997). However, a number of studies indicate that FGF8 of Mash1+ progenitors; and olfactory receptor neurons acts as a neural morphogen, which regulates expression of (ORNs), which differentiate from daughter cells of INP downstream genes that control neural patterning (Fukuchi- divisions and can be identified by expression of the neural cell Shimogori and Grove, 2001; Fukuchi-Shimogori and Grove, adhesion molecule, NCAM1 (Beites et al., 2005; Calof et al., 5212 Development 132 (23) Research article

2002; Kawauchi et al., 2004). Interestingly, the OE is one of maintained as homozygotes. Offspring were genotyped using primers the few regions of the nervous system in which neurogenesis to differentiate between flox and wild-type alleles by amplicon size and nerve cell renewal take place throughout life (Murray and (300 bp and 200 bp, respectively; F1 forward primer, 5Ј-CTTA- Calof, 1999). This capacity for continual neurogenesis GGGCTATCCAACCCATC-3Ј; F2 reverse primer, 5Ј-GGTCTC- Ј d2,3 suggests that cells within the OE produce signals that stimulate CACAATGAGCTTC-3 ). The Fgf8 (null for Fgf8 function) this process. (Meyers et al., 1998) and Foxg1-Cre (Hebert and McConnell, 2000) lines were maintained as hemizygotes (termed Fgf8d2,3/+ and In a previous study, we have shown that several FGFs, Foxg1Cre/+) on an outbred Swiss Webster (Charles River) background particularly FGF2, are potent stimulators of neurogenesis in and genotyped with specific primers: (1) Fgf8d2,3/+ (200 bp amplicon), cultured OE, where they promote divisions of OE neuronal F1 forward primer; F3 reverse primer, 5Ј-AGCTCCCG- transit amplifying progenitors and maintain the stem cells that CTGGATTCCTC-3Ј; (2) Foxg1Cre (200 bp amplicon), C1 forward give rise to these progenitors (DeHamer et al., 1994). primer, 5Ј-GCACTGATTTCGACCAGGTT-3Ј; C2 reverse primer, 5Ј- Moreover, a number of FGFs, including FGF2, are expressed GCTAACCAGCGTTTTCGTTC-3Ј. R26R mice were maintained as in and around OE at various stages of development (DeHamer homozygotes (R26RlacZ/lacZ) and genotyped using lacZ-(5Ј-CCAA- et al., 1994; Hsu et al., 2001; Kawauchi et al., 2004; Key et CTGGTCTGAGGAC-3Ј and 5ЈACCACCGCACGATAGAGATT-3Ј) al., 1996). However, two observations argue that FGF2 is or R26R locus-specific primers (Soriano, 1999). unlikely to be a crucial regulator of developmental Detection of gene expression neurogenesis in the OE. First, mice with targeted inactivation Whole-mount X-gal staining of mouse embryos was performed as of the Fgf2 gene show few if any defects in developmental described (Murray et al., 2003). For RT-PCR, E10.5 frontonasal tissue neurogenesis (Dono et al., 1998; Ortega et al., 1998). Second, (including forebrain and olfactory pit) RNA was purified using Trizol Fgf2 is not highly expressed in mouse OE until postnatal ages (Invitrogen). PCR primers were set to detect cDNA coding for Fgf8 (Hsu et al., 2001; Kawauchi et al., 2004) (S.K. and A.L.C., exon 2 and exon 3 (forward, 5Ј-GTGGAGACCGATACTTTTGG-3Ј; unpublished). Because Fgf8 has been reported to be expressed reverse, 5Ј-GCCCAAGTCCTCTGGCTGCC-3Ј). Cycling parameters in the frontonasal region near the olfactory placodes (Bachler were denaturation at 96°C for 20 seconds, annealing at 55°C for 30 and Neubuser, 2001; Crossley and Martin, 1995; Mahmood et seconds and elongation at 72°C for 1 minute, for 35 cycles. al., 1995), we hypothesized that it may serve as a signal Section in situ hybridization for E8.5-E17.5 embryos was promoting neurogenesis during early OE development. performed as described (Murray et al., 2003). For two-color in situ, one probe was synthesized using fluorescein-12-UTP and detected To test this hypothesis, we analyzed expression of Fgf8 and using AP-conjugated anti-fluorescein Fab fragments from sheep its actions on OE neurogenesis in vivo, using a conditional with INT/BCIP as the chromagen/substrate mix (Roche). For whole- genetic approach in which the Fgf8 gene was inactivated in mount in situ hybridization, E9.5-10.5 embryos were fixed overnight mouse OE from the earliest stages of OE development. Tissue in 4% paraformaldehyde in PBS with 2.5 mM EGTA at 4°C and culture assays were also used to investigate effects of processed as described (Kawauchi et al., 1999). Probes used in this recombinant FGF8 on OE neurogenesis. Expression analysis study were: ORF of mouse Fgf8 (GenBank Accession Number Development indicated that Fgf8 is initially transcribed in a small domain – MMU18673); 463 bp of mouse Fgf18 (520-983 bp of GenBank which we have termed the morphogenetic center – at the rim #AF075291); 748 bp of mouse Sox2 (1281bp-2029 bp of GenBank of the invaginating neural pit. These Fgf8-expressing cells give Accession Number X94127); and mouse Pyst1 (Dusp6 – Mouse rise to new cells that both contain Fgf8 mRNA and express Genome Informatics) (Dickinson et al., 2002). Unless otherwise indicated, Fgf8 expression was detected using a probe generated markers of OE neural stem cells. Analysis of FGF8 signaling from the full-length Fgf8 ORF cDNA (GenBank Accession and cell death demonstrate that, as a consequence of Fgf8 Number MMU18673) (Mahmood et al., 1995). Fgf8 ex1, ex2,3 inactivation, cells within the morphogenetic center and in and int probes were generated in our laboratory by PCR adjacent developing neuroepithelium undergo apoptosis. As a amplification of genomic DNA or cDNA. The int probe consists of consequence, although initial invagination of the nasal pit (NP) bp 2464-3142 of the 3274 bp intron sequence between exons 3 and and initiation of the OE neuronal lineage take place, both NP 4 (Ensemble #ENSMUST00000026241). Mash1, NgnI, Gdf11 and morphogenesis and OE neurogenesis halt shortly thereafter. Ncam1 probes were described previously (Murray et al., 2003; Wu This in turn results in a failure of development of definitive OE, et al., 2003). vomeronasal organ (VNO) – the pheromone-sensing Immunostaining and TUNEL assays component of the primary olfactory system (also derived from Cells in M-phase were detected by immunostaining using polyclonal the olfactory placode) (Farbman, 1992) – and the nasal cavity rabbit anti-phospho-histone H3 (Upstate Biotechnology, Cat. No. 06- as a whole. Thus, Fgf8 is required for the survival of cells in 570) at 1:200 dilution, visualized with Alexa Red-conjugated goat a crucial anterior morphogenetic center, which is responsible anti-rabbit-IgG (1:1000 dilution; Invitrogen). Cells in S-phase were not only for nasal cavity and OE development, but also for the detected as follows: 1 hour before sacrifice, timed-pregnant dams generation and survival of the stem cells that ultimately were given a single intraperitoneal injection of 5-bromo-2Ј- generate all cell types in the OE neuronal lineage and endow deoxyuridine (BrdU; 50 ␮g/gm body weight). Tissue was fixed and this neuroepithelium with its capacity for neuronal sectioned as for in situ hybridization, and immunostaining for BrdU regeneration. was performed as described (Murray et al., 2003). TdT-mediated dUTP nick end-labeling (TUNEL) staining to detect DNA fragmentation in apoptotic cells was performed as described (Murray Materials and methods et al., 2003), except that 20 ␮m cryosections of paraformaldehyde- fixed tissue were used and incubated with four changes of 10 mM Animals citric acid (70°C; 15 minutes per wash) following permeabilization Mice were naturally mated with the appearance of a vaginal plug and prior to the TdT reaction. Texas Red-conjugated NeutrAvidin designated as embryonic day 0.5 (E0.5). The Fgf8 allelogenic line was (1:200 dilution; Invitrogen) was used to detect incorporated biotin-16- described previously (Meyers et al., 1998). Fgf8flox/flox animals were dUTP (Roche). Fgf8 is required for olfactory neurogenesis 5213

Fig. 1. Expression of Fgf8 and neuronal cell markers in developing OE. (A) Five successive images show in situ hybridization for Fgf8 (full-length ORF probe) and OE neuronal lineage markers in invaginating nasal pit (NP) at E10.5. In whole-mount in situ hybridization, Fgf8 is detected in commissural plate and olfactory placode (white asterisk), branchial arches, mid-hindbrain junction, and limb and tail buds. Scale bar: 1 mm. In serial sections, locations of neuronal lineage markers within the OE are shown: arrowheads indicate Mash1- expressing cells, arrow indicates Ncam1- expressing neurons. Scale bar: 200 ␮m. (B) Double label in situ hybridization for Fgf8 (full-length ORF probe, orange) and Sox2 (blue) demonstrates overlap of the two markers in a small rim of surface ectoderm and adjacent invaginating neuroepithelium (brackets). Scale bar: 50 ␮m. (C) In situ hybridization for unprocessed, intronic RNA (Fgf8int probe) expression and processed Fgf8 mRNA (Fgf8ex2,3 probe) expression. Red and blue arrowheads indicate the basolateral extent of intronic RNA versus processed mRNA expression, respectively. The domain of mRNA expression subsumes that of intronic expression. Scale bars: 50 ␮m. LNP, lateral nasal process; MNP, medial nasal process. (D) Model of peripheral-to-central process of neuronal differentiation in developing OE and origin of Sox2-expressing neural stem cells from Fgf8-expressing ectoderm.

Development Results experiments to try to determine if a subpopulation of the Fgf8- Fgf8 is expressed in a neurogenic domain of expressing cells in the NP are early OE neural stem cells. In invaginating nasal epithelium the first experiment, we used double-label in situ hybridization To understand how Fgf8 might act to regulate OE for Fgf8 (ORF probe) and Sox2 on the same sections. The neurogenesis, we first performed in situ hybridization for Fgf8 results, shown in Fig. 1B, indicate that many Fgf8-expressing [using a probe encompassing the entire open reading frame cells that lie within the neuroepithelium of the invaginating NP (ORF) of the cDNA; see Materials and methods] and OE also express Sox2. These cells also express Pax6 and Dlx5, neuronal lineage markers on serial sections of invaginating NPs other markers of definitive OE at this stage (see Fig. S1 in the at day 10 of gestation (E10.5). As shown in Fig. 1A, Fgf8 is supplementary material). Thus, by the criterion of Sox2 expressed in cells within a domain that encompasses a ring of expression, a subpopulation of Fgf8-expressing cells can be ectodermal epithelium at the rim of the invaginating NP, as well considered to be early OE neural stem cells. The presence of as adjacent neuroepithelial cells inside the pit. Sox2 expression cells that co-express both Fgf8 and Sox2 suggested to us that is observed throughout the entire neuroepithelium of the Fgf8 expression defines a region from which neural stem cells invaginating NP, and defines the OE at this early stage. emerge and enter the invaginating olfactory neuroepithelium. Interestingly, expression of different OE neuronal cell type- Moreover, the sequential appearance of cells expressing specific markers occurs in an outside-in pattern that reflects the markers of successively more differentiated stages in the OE stage of each expressing cell in the neuronal lineage: cells neuronal lineage, in NP regions that are further and further expressing Mash1, which marks the earliest committed from the Fgf8-expressing domain, is consistent with the idea neuronal progenitors, are present next to the Fgf8-expressing that OE morphogenesis and initiation of the OE neuronal cells at the inner rim of the NP; adjacent to Mash1-expressing lineage proceed in an outside-in fashion, with early stem cells cells are Ngn1-expressing INPs; and in the center of the pit lie at the periphery and terminally differentiated ORNs in the cells that are positive for Ncam1 (expressed by postmitotic center of the NP (cf. Cau et al., 1997). ORNs). In a second set of experiments, we investigated the origin Because the data in Fig. 1A suggest an overlap in the of Fgf8-expressing cells in the OE neuroepithelium, using a expression domains of Fgf8 and Sox2, which marks many early technique similar to that of Dubrulle and colleagues neuroepithelial stem cells as well as OE neural stem cells once (Dubrulle and Pourquie, 2004). In situ hybridization was the definitive OE structure has been established (Beites et al., performed on adjacent sections of invaginating NP, in one 2005; Ellis et al., 2004; Graham et al., 2003; Kawauchi et al., case using a probe to intron sequences to determine the 2004; Wood and Episkopou, 1999), we performed two sets of location of cells that initially transcribe unprocessed Fgf8 5214 Development 132 (23) Research article

RNA (‘Fgf8 int’ probe), and in the second case a probe for Localization of Foxg1-driven Cre activity and Fgf8 exons 2 and 3 of the processed mRNA (‘Fgf8 ex2,3’ probe). conditional knockout strategy The results, shown in Fig. 1C, demonstrate that the cells To examine the functional importance of our observations from which initially transcribe Fgf8 form a subset of all cells that in situ hybridization and tissue-culture studies, we used a actually contain Fgf8 RNA in this region. These Fgf8- conditional Fgf8 knockout model, using Cre-LoxP tissue- transcribing cells appear to be located preferentially at the specific gene disruption to determine the role of FGF8 rim of the NP and in the basal region of the invaginating signaling in OE development from E10.5 to birth [complete neuroepithelium. By contrast, cells that contain processed loss of Fgf8 results in early embryonic lethality (Meyers et al., mRNA for Fgf8 (positive for the Fgf8 ex2,3 probe) are found 1998; Sun et al., 1999)]. A transgenic mouse line in which Cre more extensively both within invaginating neuroepithelium recombinase expression is controlled by Foxg1 (forkhead box and in the ectoderm surrounding the NP. As cells that are Fgf8 G1, also known as brain factor 1; Foxg1-Cre line) regulatory ex2,3 positive must be the progeny of the Fgf8 int-positive elements was chosen, as mice carrying this allele have been cells (Dubrulle and Pourquie, 2004), these findings suggest a shown to express Cre in anterior neural structures, including lineal relationship between the Fgf8-expressing ectodermal developing OE and forebrain (Hebert and McConnell, 2000). cells that define the rim of the invaginating NP and the To confirm Cre activity in vivo, we crossed ROSA26 reporter neuroepithelial cells that express both Fgf8 mRNA and Sox2. mice [R26R (Soriano, 1999)] with Foxg1-Cre mice and Thus, at least some of the Sox2-expressing neural stem cells analyzed resulting progeny by X-gal staining. Clear staining in the developing OE must be derived from Fgf8-expressing was observed in the anterior neural ridge at E8.5, prior to the ectodermal cells. Altogether, these observations suggest that time that olfactory placodes form (data not shown). A day later, Fgf8 expression defines a morphogenetic center that gives at E9.5, we observed staining in olfactory placodes and OE, as rise to at least some of the Sox2-expressing neural stem cells well as forebrain neuroepithelium and branchial arches; OE of the OE, and that the earliest of these neural stem cells and forebrain structures continued to be stained at later times themselves transcribe Fgf8. These ideas are depicted in the in development (Fig. 2A). These data indicate that the Foxg1- cartoon shown in Fig. 1D. Cre line is suitable for driving tissue-specific expression of Cre At later stages (beyond E12-13), OE stem and progenitor in olfactory neuroepithelium from the earliest time at which it cells take on a more restricted location, and come to lie in a can be observed to be differentiating from head ectoderm. compartment adjacent to the basal lamina of the epithelium The strategy for generating conditional mutants is outlined (Kawauchi et al., 2004). Analyses by in situ hybridization and in Fig. 2B. Fgf8flox/flox;Foxg1+/+ females were crossed with RT-PCR indicate that Fgf8 continues to be expressed in Fgf8d2,3/+;Foxg1Cre/+ males, and ~25% of embryos generated scattered cells located in the basal compartment of OE at were Fgf8flox/d2,3;Foxg1Cre/+ animals (hereafter referred to as E14.5 (see Fig. S2 in the supplementary material). These data ‘mutant’ animals). Mutant animals were detected at all suggest that Fgf8 continues to be expressed in or near stem embryonic stages and in newborn litters, but died shortly after Development and progenitor cells of the OE. Moreover, in vitro assays birth owing to multiple defects (see below). show that recombinant FGF8 is capable of stimulating development of neural stem cells and proliferation of Mutant embryos have severe defects in frontonasal neuronal progenitors in cultures taken from E14.5 OE (see structures Fig. S3 in the supplementary material), demonstrating that Intact mutant embryos were examined from E9.5 to birth (Fig. OE progenitors are responsive to FGF8 at this stage of 3A). Defects are evident as early as E9.5, primarily as a development. reduction in the size of the forebrain and frontonasal structures.

Fig. 2. Localization of Foxg1-driven Cre expression and strategy for generating Fgf8 conditional knockouts. (A) X-gal staining of R26RlacZ/+;Foxg1Cre/+ embryos at E9.5, E10.5 and E12.5. Top panels show whole-mount images and bottom panels show corresponding sections from embryos processed as whole mounts. At E9.5, staining is detected in forebrain (FB) and olfactory placode (arrows). At E10.5, staining is observed at rim of invaginating nasal pit (NP; arrows). OC, optic cup. At E12.5, prominent staining is observed in OE. Scale bars: 500 ␮m in the top panels; 200 ␮m in the bottom panels. (B) Fgf8flox/flox,Foxg1+/+ females were crossed with Fgf8d2,3/+;Foxg1Cre/+ males and embryos genotyped with three different primer sets to detect different alleles: Fgf8-flox and wild-type (F1 and F2), Fgf8-d2,3 (F1 and F3) and Cre (C1 and C2). One-quarter of offspring had the Fgf8d2,3/flox;Foxg1Cre/+ (mutant) genotype. Fgf8 is required for olfactory neurogenesis 5215

Fig. 3. Deficits in anterior neural structures and FGF- mediated signaling in mutant embryos. (A) Pictures of mutant animals and control littermates from E9.5 to birth. White arrowheads indicate forebrain-midbrain boundary. Black asterisks indicate nasal pits in E10.5 mutants. P, pinna. Scale bars: 1 mm. (B) Whole- mount in situ hybridization with Fgf8 exon2,3 probe in E10.5 embryos. White arrows indicate nasal pits. CP, commissural plate. Insets show forelimbs of embryos hybridized with Fgf8 ex2,3 probe. Scale bar: 0.5 mm. (C) RT-PCR for Fgf8 exons 2 and 3 using cDNA from E10.5 forebrain/frontonasal tissue of control and mutant animals. Lane 1, 100 bp marker; lane 2, control tissue with Fgf8 ex2,3 primers; lane 3, control tissue and no RT; lane 4, mutant tissue with Fgf8 ex2,3 primers; lane 5, mutant tissue with no RT; lane 6, Fgf8 exon2,3 plasmid control; lane 7, control cDNA and Hprt primers; lane 8, mutant cDNA with Hprt primers. (D) Pyst1 expression is reduced in OE and underlying mesenchyme (arrows in insets). R, retina; FB, forebrain. No Development change in Shh expression is apparent in mutants. MB, midbrain. Scale bar: 200 ␮m.

Strikingly, all mutant animals have at least a rudimentary frontonasal tissue of E10.5 embryos using primers specific for olfactory pit at E10.5; however, by this time, defects in medial the exon 2,3-coding region. Expression was readily detectable nasal process development are often apparent, with this region in tissue from control animals, but was undetectable in mutants being flattened and smaller than in controls. From E12.5 (Fig. 3C). Together, these data confirm that mutants express no onwards, the forebrains of mutant embryos show dramatic detectable Fgf8 mRNA in the frontonasal region. reductions in size compared with control littermates; limbs and other posterior structures appear grossly normal, however. At FGF signaling is downregulated in Fgf8 mutant birth, mutants have a small short snout, and the lower jaw, embryos pinnae (outer ears) and eyelids are either reduced in size or Experiments described above demonstrate that Fgf8 gene absent. The size and gross structure of the eye itself appear to expression is effectively eliminated in the OE of mutant be unaffected in mutants (data not shown). Littermate embryos animals. However, it might still be the case that FGF8- with genotypes other than Fgf8flox/d2,3;Foxg1Cre/+ showed mediated signaling could somehow be compensated for in normal development and survived to adulthood. mutants, thus complicating analysis of phenotypes. To test this To confirm that the phenotype we observed is due to absence hypothesis, we examined expression of Pyst1/Mkp3 in the of Fgf8, we performed whole-mount in situ hybridization (Fgf8 frontonasal region of E10.5 mutant and control littermate ex2,3 probe) and RT-PCR at E10.5 to ensure that mutant embryos. Pyst1 encodes a tyrosine phosphatase that is an FGF- embryos no longer express exons 2 and 3 of Fgf8, the regions inducible antagonist of FGF signaling (Eblaghie et al., 2003), of the gene that should be excised by Cre recombinase in and is one of a number of genes in the FGF ‘synexpression’ mutant animals. In controls, Fgf8 ex2,3 expression was group, i.e. genes expressed in the same temporal and spatial detected in olfactory pits, commissural plate and branchial pattern when FGFs initiate signaling (Niehrs and Meinhardt, arches, whereas no signal was detected in these regions in 2002). Pyst1 is expressed in many known sites of FGF mutants (Fig. 3B). Importantly, the Fgf8 ex2,3 probe could signaling in mouse embryos, including the olfactory system detect normal Fgf8 expression in the apical ectodermal ridge (Dickinson et al., 2002), making its expression useful as a read- of the limb in both mutant and control embryos (Fig. 3B, out for active FGF signaling. As shown in Fig. 3D, Pyst1 is insets). To ensure that no residual active Fgf8 mRNA is normally expressed in the rim of the invaginating olfactory pit produced in mutant animals, we also performed RT-PCR on (where Fgf8 itself is expressed) and in the mesenchyme 5216 Development 132 (23) Research article

throughout the medial nasal process. In mutant embryos, examined at each age, with a total of 31 analyzed). Normal OE expression of Pyst1 is strongly downregulated in both domains development is shown in Fig. 4A. At E10.5, some olfactory (insets), confirming that loss of Fgf8 leads to severe placode/nasal pit structure was observed in all animals, even decrements in FGF signaling in the developing olfactory mutants; however, the OE and nasal cavity were severely region. reduced in size or absent in all mutant embryos by E11.5 (Fig. We also examined expression of Shh, which, like Fgf8, is a 4B). On the basis of histology, mutants were placed into one key signaling molecule in limb and telencephalon (Ohkubo et of two categories: Type A (aplastic) mutants had essentially no al., 2002; Panman and Zeller, 2003), and which evidence nasal cavity or OE detectable from E11.5 onward; Type B suggests may be positively regulated by FGFs (Niswander et (hypoplastic) mutants retained a vestige of nasal cavity, usually al., 1994; Zuniga et al., 1999). As shown in Fig. 3D, Shh is not present as an S-shaped tubular structure lined with an expressed in OE at E10.5, but instead is expressed in the ventral epithelium, at E11.5 and after. The numbers of each type of wall of the telencephalon next to the medial nasal process and mutant found at each age are given in Table 1. Interestingly, at forebrain commissural plate. In mutants, the basic pattern of E10.5 some mutant embryos already displayed a phenotype, in Shh expression appears unaffected. Expression of the SHH that they had extremely small placodes and obvious reductions receptor patched, which is autoregulated through SHH in the sizes of the lateral and medial nasal processes (Fig. 4B). signaling in overlapping and complementary patterns, also We surmise that such embryos would exhibit the more severe, appeared unaffected in mutants (data not shown) aplastic OE phenotype at later stages. Nasal bone structures are (Drossopoulou et al., 2000; Marigo and Tabin, 1996; Platt et present in hypoplastic (Type B) mutants, and closely surround al., 1997). These observations indicate that effects on any remnant of OE, suggesting that bone formation in this neurogenesis observed in mutants are unlikely to be mediated region is at least partially dependent on OE development, indirectly via Shh signaling (LaMantia et al., 2000). possibly through an inductive signal derived from OE tissue. Of significance also is the fact that no vomeronasal organ Severe reduction or absence of olfactory structures (VNO) structure was observed in any mutant animals, whether in mutant embryos these exhibited aplastic or hypoplastic phenotypes. As the To evaluate OE formation and growth in the absence of Fgf8, VNO is derived from the olfactory placode, and starts to we analyzed sections of mutant and control animals from develop during the period when olfactory pits are invaginating E10.5 to E17.5 (a minimum of four mutant animals were (around E11.5) (Farbman, 1992), this observation indicates an absolute requirement for Fgf8 in VNO formation. Neuronal cells form initially in mutant OE but fail to increase

Development in number The phenotypes observed in mutant animals suggested that Fgf8 would be likely to affect development of cells in the OE neuronal lineage. To test this, we used in situ hybridization to compare expression of neuronal lineage markers in the OE of mutants and controls at four ages spanning the extent of prenatal OE development. Fig. 5A shows that at E10.5, mutant OE, which does not express functional Fgf8, continues to express Pax6, a gene known to be important in early determination of the OE

Fig. 4. Histological analysis of mutant OE. (A) Schematic model of mouse OE development. OP, olfactory placode; NP, nasal pit; FB, forebrain; MNP, medial nasal process; LNP, lateral nasal process; S, nasal septum; NC, nasal cavity. (B) Hematoxylin-eosin staining was performed on 20 ␮m cryosections through the entire frontonasal region of control and mutant embryos from E10.5- E17.5. Scale bar: 200 ␮m. CP, cartilage primordium of nasal capsule; H, heart; L, lens; MB, midbrain; NR, neural retina; Tel, telencephalon. Fgf8 is required for olfactory neurogenesis 5217

Table 1. Phenotypes of Fgf8 conditional mutants Table 2. Deficits in neuronal cell types in the OE of Fgf8 Type A Type B mutants at E10.5 Gestational Aplastic OE Hypoplastic OE Total embryos Mean number of cells/ age (% of total embryos) (% of total embryos) examined Neuronal 2 lineage 0.03 mm area (s.e.m.) n (nasal pits E10.5 0 (0%) 7 (100%) 7 marker Mutant Control evaluated) P (t-test) E11.5 2 (40%) 3 (60%) 5 E12.5 2 (29%) 5 (71%) 7 Mash1 4.45 (1.16) 23.30 (3.47) Mutant=4 0.002 E14.5 3 (37%) 5 (63%) 8 Control=4 E17.5 3 (75%) 1 (25%) 4 Ngn1 33.37 (3.02) 48.57 (1.28) Mutant=6 0.012 Control=3 Each embryo was cryosectioned (20 ␮m) throughout the entire frontonasal region and sections stained with Hematoxylin-eosin. Criteria for Ncam1 7.29 (2.58) 23.95 (3.98) Mutant=3 0.025 categorization of mutants is discussed in the text. Control=3 In situ hybridization for each neuronal marker was performed on serial sections (20 ␮m) through the full extent of nasal pits in e10.5 mutants and (Grindley et al., 1995). In addition, neuronal lineage markers littermate controls (20-25 sections in controls, 15-20 sections in mutants). in E10.5 mutants are expressed in a pattern generally similar The number of cells expressing a given marker and total OE area were to that seen in controls: the early progenitor marker Mash1 is measured for all sections encompassed by a given nasal pit. For comparison, 2 expressed peripherally, with Ngn1 expressed central to Mash1 cell counts were normalized to an area of 0.03 mm , the average area of OE in a single section. and Ncam1 expressed in the deepest recess of the invaginating pit (Fig. 5A). We were surprised at this finding, as the OE is so severely affected in mutants at ages greater than E10.5. To largest reduction in Mash1-expressing cells [the first determine if some defect in neurogenesis is already present at committed progenitor cells in the neuronal lineage (Calof et E10.5, we counted cells expressing each lineage marker al., 2002)]: a greater than fivefold reduction was seen in the throughout the entire extent of the nasal pit in mutant embryos number of these cells in mutant OE, compared with controls. and control littermates at this age. The data are shown in Table Thus, despite the fact that all cell types in the OE neuronal 2. Interestingly, every neuronal cell type is affected, with the lineage initially form, major deficits in the numbers of these Development

Fig. 5. Cessation of neuronal lineage development in mutant OE. (A) In situ hybridization on serial coronal sections through E10.5 nasal pit. No Fgf8 ex2,3 is detected in mutant. (B) In situ hybridization on serial coronal sections of E12.5 Type A mutant. Arrowhead indicates region of Sox2-expressing epithelium in sections hybridized with other probes. (C) In situ hybridization on horizontal serial sections of E14.5 Type B mutant. Arrow indicates apparent OE remnant. (D) In situ hybridization on horizontal sections of E17.5 Type B mutant. Arrowheads indicate presumptive remnant of OE. NP, nasal pit; MNP, medial nasal process; VG, Vth ganglion; OC, oral cavity; T, tongue; VNO, vomeronasal organ; FB, (presumptive) forebrain; NR, neural retina; S, nasal septum. Scale bars: 200 ␮m. 5218 Development 132 (23) Research article

cells are already apparent at E10.5, indicating that OE numbers of apoptotic cells were observed throughout the Fgf8- neurogenesis is already severely affected by absence of Fgf8 expressing domain in mutants (detected in adjacent sections at this age. using a probe to Fgf8 exon 1, which is expressed, but does not In analyses of older animals, we used an in situ hybridization generate functional protein, in Fgf8 mutants). By contrast, very probe for Sox2 to delineate potential remnants of OE in the few apoptotic cells were observed in the Fgf8-expressing frontonasal regions of mutants. An example of a Type A domain (detected using the Fgf8 exon2,3 probe) in control (aplastic) mutant at E12.5 is shown in Fig. 5B. In Type A littermates. The increase in the number of TUNEL-positive mutants, frontonasal structures are drastically reduced in size, cells was greatest at E10.5, when mutant animals showed a 37- resulting in compression of dorsal and ventral structures such fold increase compared with controls [mutant: 1220±485 that no obvious nasal cavity can be seen. In this particular (s.e.m.); control: 37±18 (s.e.m.)]. At E12.5, the difference embryo, a region of Sox2-expressing epithelium (Fig. 5B, between mutant and control mice was smaller but still arrowhead) was observed to bud off from the oral cavity (OC), significant (Fig. 6B-C), whereas by E14.5 cell death was low, suggesting that this might be a remnant of nasal cavity and and had decreased to approximately the same level in mutants contain OE neuronal cells. However, as shown in adjacent as in controls (Fig. 6C). Interestingly, Sox2 expression also sections hybridized with probes for specific lineage markers, appears to be attenuated in the OE regions, showing high levels no neuronal cells were present in this region [the patches of of apoptosis in mutants, particularly the OE lining the NP in ectopic Mash1-expressing cells in Fig. 5B (mutant) are not the medial nasal process (Fig. 6A, Sox2 panel, arrowhead), within the Sox2-expressing epithelium, and are probably where FGF8 signaling is strongly reduced in mutants (Fig. 3D). sections through cranial ganglia]. Thus, by E12.5, neuronal To determine if absence of functional Fgf8 results in a development in the OE appears to have ceased altogether in change in cell proliferation, we performed immunostaining for Type A mutants. the M-phase specific marker, phosphorylated histone H3 (Galli Fig. 5C,D show Type B (hypoplastic) mutants, which retain et al., 2004), and for BrdU incorporation (which detects cells small vestiges of OE. Even in mutants with this less severe in S phase), at E10.5. M-phase cells were observed primarily phenotype, almost no OE neuronal cells remain, and the OE in the surface (apical) layer of OE in both control and mutant itself is very small compared with that of controls. animals, but there was no obvious difference in either the Interestingly, we detected expression of Fgf18, which is closely pattern or number of immunopositive cells (Fig. 6D). To related to Fgf8 in structure and function (Maruoka et al., 1998; confirm this, total numbers of phosphohistone H3- Ornitz and Itoh, 2001), in the OE remnant present in an E14.5 immunopositive cells were counted in serial sections through Type B mutant (Fig. 5C, arrows). This observation suggests the entire extent of the nasal pits in mutant and control animals. that, in Type B mutants, there may be some compensation for The results, shown in Fig. 6D,E confirm that there is no loss of Fgf8 function by Fgf18, provided that the OE is able to significant change in the mitotic index of OE cells in the develop to the stage when Fgf18 normally starts to be absence of Fgf8. Levels of BrdU immunostaining were high in Development expressed in this region (about E12.5) (S. Kawauchi, data not OE in both mutants and controls, and BrdU+ cells were shown) (see Bachler and Neubuser, 2001; Xu et al., 2000). Fig. particularly dense in the basal half of the epithelium in both 5D illustrates that, in older Type B mutants (E17.5), even when cases (Fig. 6F). However, quantification of these sections again a relatively large remnant of nasal cavity persists (arrowheads), demonstrated that there was no significant decrease in the the epithelium that lines it is essentially devoid of cells number of BrdU-immunopositive cells (i.e. cells in S phase) in expressing genes specific to OE neuronal progenitors and the OE of mutant animals, compared with control littermates ORNs (Mash1, Gdf11, Ncam1) (see Wu et al., 2003). These (Fig. 6G). Thus, the changes in OE neurogenesis seen at E10.5 observations indicate that, whatever process goes awry in Fgf8 appear to be the result of increased cell death, not decreased mutant OE, deficits appear very early in development, and the cell proliferation. effects of these deficits are long lasting, such that few if any neuronal cells are able to form. Discussion Loss of Fgf8 results in increased cell death, not Although other investigators have noted expression of Fgf8 in decreased cell proliferation developing olfactory pit (Bachler and Neubuser, 2001; By what mechanism does loss of Fgf8 cause loss of neuronal Crossley and Martin, 1995; Mahmood et al., 1995), as well as stem and progenitor cells in the OE? Potentially, FGF8 could some effects on OE development in vitro (LaMantia et al., stimulate neurogenesis by promoting proliferation and/or 2000; Shou et al., 2000), the present study is the first to survival of neuronal stem and progenitor cells. Indeed, as demonstrate that Fgf8 function is required for development of described above, recombinant FGF8 – like other FGFs – is able the OE and maintenance of neurogenesis in this tissue in vivo. to promote development of OE stem cells and proliferation of Fgf8 is first expressed within a peripheral domain of INPs in tissue culture assays (see Fig. S3 in the supplementary ectodermal cells at the rim of the invaginating nasal pit, which material) (DeHamer et al., 1994; Shou et al., 2000). To we have termed a morphogenetic center. Our data indicate that determine the mechanism(s) by which Fgf8 acts in vivo, we the cells that initially transcribe Fgf8 give rise to other cells performed in situ assays for both apoptotic cells and that contain processed Fgf8 mRNA and in addition express the proliferating cells in mutant and control animals at early stages neural stem cell marker Sox2. From these observations, we of NP invagination and OE development. conclude that at least some of the Sox2-expressing neural stem To identify apoptotic cells, we performed TUNEL assays on cells of the OE are derived from the Fgf8-expressing cryosections of OE of mutants and control littermates at E10.5, ectodermal cells of the morphogenetic center that rims the E12.5 and E14.5. The results are shown in Fig. 6A-C. Large invaginating nasal pit. In the absence of Fgf8 function, there is Fgf8 is required for olfactory neurogenesis 5219

Fig. 6. Fgf8 is required for cell survival in the neurogenic domain. (A) In situ hybridization for Fgf8 ex1 probe indicates areas where (nonfunctional) Fgf8 is expressed in mutants (Fgf8 ex2,3 probe signal was not detected in mutants). TUNEL panel shows high number of apoptotic cells in mutants in ectoderm (white asterisk) and OE (white arrowhead; magnified in inset) of invaginating nasal pit (NP). Hoechst panel shows extent of invaginating NP. LNP, lateral nasal process; MNP, medial nasal process. Sox2 expression is reduced in OE of MNP (black arrowhead). Scale bars: 100 ␮m. (B) High-power micrographs of TUNEL staining in OE of E12.5 Type B mutant and control littermate. Broken white line indicates basal lamina (BL) of OE. AL, apical surface; Str, stroma. Scale bar: 50 ␮m. (C) Total TUNEL+ cells in identifiable nasal epithelium (NE) were counted, and area of NE measured, in multiple sections at each age indicated. Data for each NP were summed and normalized to 0.1 mm2, the average total NE area in a section at E12.5. Mean±s.d. for data from individual NPs are shown; NPs from a minimum of 2 animals of each genotype at each age were counted. Differences between mutants and controls were statistically significant at E10.5 (P=0.009, Student’s t- test) and E12.5 (P=0.003), but not E14.5. (D) High- power micrographs of anti-phosopho-Histone H3 immunostaining at E10.5. Scale bar: 50 ␮m. (E) Quantification of data illustrated in D. (F) High- power micrographs of anti-BrdU immunostaining at E10.5. Scale bar: 50 ␮m. (G) Quantification of data illustrated in F. There are no significant differences between datasets in E and G . Data for each NP were summed and normalized to 0.03 mm2, the average total OE area in a section at E10.5. Mean±s.d. for data from individual NPs is shown; NPs from a minimum of two

Development animals of each genotype at each age were counted.

a large reduction in FGF-mediated signaling and a wave of apoptosis in cells within the Fgf8- expressing domain; this also results in a reduction in the number of Sox2-expressing putative neural stem cells. This wave of apoptosis, which begins at the earliest stages of OE development and persists for at least 2 days, severely limits subsequent expansion of the OE and associated neurogenesis. Nasal cavity development is profoundly affected and VNO structures never form. These findings, summarized in Fig. 7, demonstrate that Fgf8 is a crucial determinant of OE and VNO development, and nasal cavity morphogenesis, and is required for olfactory neurogenesis. In addition, they which first appear as two oval epithelial patches in the indicate that cells in the Fgf8-expressing domain are crucial for anterolateral region of the head around E9 in the mouse the generation and survival of the stem cells that ultimately (Farbman, 1992). One day later, the placodes invaginate to generate all cell types in the OE neuronal lineage, and indicate form the nasal pits (NPs), which continue to deepen and that some neural stem cells must ultimately be derived from elaborate a stereotyped pattern of folds (nasal turbinates) as Fgf8-expresing cells. Thus, when Fgf8 is absent, neuronal development proceeds. The characteristic morphology of the progenitor cell populations cannot be replenished and nasal cavity and distribution of different cell types within the neurogenesis ultimately ceases. OE emerge at about E13-14. At this time, the overall number of mitotic figures decreases, and proliferating cells begin to Fgf8 and the establishment of the OE neuronal concentrate in the basal layers of the OE, where neuronal lineage progenitors and stem cells ultimately reside (Cuschieri and The cells of the OE originate from the olfactory placodes, Bannister, 1975; Smart, 1971). These changes in cell 5220 Development 132 (23) Research article

Fig. 7. Role of Fgf8 in olfactory neurogenesis. Cartoon illustrating the relative positions of the Fgf8 expression domain (anterior morphogenetic center) and different neuronal cell types during primary neurogenesis at E10.5 in normal OE (wild type) and a model for the role of Fgf8 in primary olfactory neurogenesis based on the consequences of inactivating Fgf8 in the anterior morphogenetic center using Foxg1-driven Cre (mutant). Fgf8 expression domain, orange; Sox2 expression domain (definitive neuroepithelium), yellow; primordial neural stem cells (co-expressing Sox2 and Fgf8), green; Mash1-expressing committed neuronal progenitors, dark blue; INPs, light blue; Ncam1-expressing neurons, pink. Cells in the Fgf8 expression domain that undergo apoptosis when Fgf8 is inactivated are shown in red, and apoptotic primordial neural stem cells in green with a red jagged border. Vestigial populations of other neuronal cell types are shown in their corresponding colors, but with jagged borders. For discussion, see text.

proliferation and location that occur at this time appear to mark mutants as late as E14.5 (Fig. 5). However, as neuronal stem a developmental transition from an early morphogenetic form cells ultimately die in the absence of Fgf8, the mature of neurogenesis (primary neurogenesis) to neurogenesis of an characteristics of the OE and neurogenesis within this established pattern, in which the proportions of different cell epithelium are never established, even in the least severe types are maintained at a fairly constant level within the OE mutants (Fig. 5). These findings are consistent with (established neurogenesis) (see Kawauchi et al., 2004). observations made in studies of various Fgf8 mutants in other What role does Fgf8 play in establishing the OE neuronal tissues. For example, when Fgf8 expression is eliminated from lineage and in primary neurogenesis? Our findings indicate that the apical ectodermal ridge (AER) of developing limbs, AER Fgf8 is initially transcribed in the most peripheral domain of cells and the mesenchyme that underlies them undergo Development the invaginating NP, outside of the region where lineage- apoptosis (Sun et al., 2002). In addition, Storm and colleagues specific markers such as Mash1 and Ngn1 are expressed (Fig. have reported that telencephalic neural progenitors undergo 1). The Fgf8 mRNA expression domain expands and comes to apoptosis when Fgf8 is eliminated, the likely cause of the include neuroepithelial cells that express genes that are defects in forebrain development observed in our study (Storm definitive markers of olfactory neuroepithelium, including et al., 2003) (compare with Fig. 3). Our results, in addition to Sox2, Dlx5 and Pax6 (Fig. 1 and see Fig. S1 in supplementary these and results from a number of labs studying Fgf8 function, material). Indeed, recent data suggest that Sox2 expression suggest that FGF8 acts as a survival factor for crucial stem cell characterizes the stem cell population in the OE during populations in a wide variety of tissues in which it is expressed established neurogenesis (Kawauchi et al., 2004; Beites, 2005). (Abu-Issa et al., 2002; Chi et al., 2003; Frank et al., 2002; Our observations therefore suggest that Fgf8 is expressed in a Storm et al., 2003; Sun et al., 2002; Trumpp et al., 1999). morphogenetic center, in and around the cells that are Although the molecular details of how Fgf8 prevents cell responsible for initiating primary olfactory neurogenesis – death are incompletely understood, our analysis of Pyst1, cells that we now consider to be the primordial neural stem which encodes a MAPK-specific phosphatase whose cells of the OE, by analogy to the primordial germ cells that expression is dependent on MAPK signaling (Eblaghie et al., ultimately give rise to gametes. In the absence of Fgf8 function, 2003), indicate that the RAS/MAPK pathway activity may be severe deficits in primary neurogenesis rapidly take place (Figs involved. This pathway is known to regulate apoptosis in other 3-5) owing to the high levels of apoptosis that take place in the systems (Downward, 1998). One possibility, suggested by our Fgf8 expression domain (Fig. 6). This model of Fgf8 action data, is that absence of Fgf8 at this early crucial juncture leads during primary OE neurogenesis is depicted in Fig. 7. to downregulation of the RAS/MAPK pathway, which ultimately acts as a trigger for death of the primordial neural Fgf8 acts by controlling cell survival stem cells that are ultimately responsible for establishing the A dramatic phenotype observed in Fgf8 mutants was the very neurogenic pathway in the OE. Consistent with this notion, in high level of programmed cell death at E10.5 (Fig. 6). In vitro studies have shown a relationship between maintenance mutants at this time, cells in the Fgf8-expressing domain, as of FGF signaling and prevention of apoptosis in P19 cells well as surrounding neuroepithelium, are unable to survive and (Miho et al., 1999). the numbers of all neuronal cell types are subsequently depleted. The few cells already committed to the neuronal Are effects of loss of Fgf8 direct or indirect? lineage appear to be able to continue through the maturation Overall, our observations suggest a model in which FGF8 acts process, as some neuronal cells can be observed in Type B in an autocrine and/or paracrine manner in cells of the Fgf8 is required for olfactory neurogenesis 5221

developing OE and surrounding anterior ectoderm. However, Fgf8 and Sox2 expression (Fig. 1B), these findings strongly the dramatic effects of loss of Fgf8 function on both suggest that at least some Fgf8-expressing cells become what craniofacial morphogenesis and forebrain development, we have termed primordial neural stem cells of the OE observed by us in the present study and by others in this and (Fgf8+/Sox2+ cells) [compare Fig. 7 with Kawauchi et al. other Fgf8 mutants (Abu-Issa et al., 2002; Storm et al., 2003; (Kawauchi et al., 2004)]. Therefore, as apoptosis in mutant OE Trumpp et al., 1999), raise the alternative possibility that is most extensive in the Fgf8+ domain (Fig. 6B), we infer that effects on OE neurogenesis observed in mutants could be at least some of the cells that are dying in mutants are caused indirectly, owing to effects on these other tissues. As primordial neural stem cells, an idea supported by the finding we observe a reduction is FGF-mediated signaling in the that Sox2 expression is attenuated in mutant OE in the region mesenchyme surrounding the invaginating olfactory where Fgf8 expression and Sox2 expression normally overlap neuroepithelium, as well as in the Fgf8-expressing (Fig. 6A). However, as the regions of both Fgf8 expression neuroepithelium itself (Fig. 3C), the possibility that loss of and apoptosis in the mutant extend beyond the neuroepithelial Fgf8 disrupts an epithelial-mesenchymal signaling loop that domain defined by Sox2 expression, it is also likely to be the may feed back to promote OE neurogenesis cannot be case that some of the dying cells in mutants are not committed discounted totally by our data. Because by far the most cell neural stem cells. Thus, we conclude that apoptotic cells death we observe in mutants is in Fgf8-expressing ectoderm include both Fgf8-expressing cells committed to the neural and neuroepithelium, and not in underlying mesenchyme (Fig. lineage of the OE (i.e. primordial neural stem cells), as well 6B and data not shown), we do not think that alterations in Fgf8-expressing cells that may not be destined to undergo this FGF8-mediated signaling in mesenchyme are responsible for commitment step. Ultimately, the decreased numbers of all the defects – especially cell death – that we observe in OE. It neuronal cell types, the failure of the OE and the VNO to may still be the case that loss of FGF8 signaling in develop, and abortive nasal cavity morphogenesis in mutant mesenchyme contributes to the defects in nasal cavity animals, together demonstrate the profound dependence of formation we observe, however, possibly by interrupting BMP- both OE neurogenesis and anterior craniofacial development mediated signaling in this tissue; we are currently exploring on developmental expression of Fgf8. this possibility (S.K., C. E. Crocker and A.L.C., unpublished). Because Fgf8 mutants also exhibit strong defects in the We thank Felix Grün, Arthur Lander and Calof laboratory members developing telencephalon, it might also be argued that proper for discussions; Craig MacArthur, David Ornitz, Elizabeth Robertson, OE development is dependent on proper formation of the John Rubenstein, Robin Dickinson and Peter Gruss for reagents and olfactory bulbs (OBs), and thus that effects on OE probes; and Erik Meyers for Fgf8 allelogenic mouse lines. This work was supported by grants to A.L.C. from the NIH (DC03583 and neurogenesis in mutants might be indirectly mediated via HD38761) and the March of Dimes. S.K. was a Long-Term Fellow effects on the OB. To test this idea, we examined OE of the Human Frontier Science Program, and R.S. was supported by development and OB structure using in situ hybridization in the UCI NIH Minority Biomedical Research Support Program. Development Fgf8 hypomorphs (Fgf8neo/neo), which have been reported to lack most or all OB tissue (Garel et al., 2003; Meyers et al., Supplementary material 1998). Our observations indicate that the normal complement Supplementary material for this article is available at of Ncam1-expressing ORNs is present in the OE of Fgf8 http://dev.biologists.org/cgi/content/full/132/23/5211/DC1 hypomorphs at P0, even though these animals fail to develop any proper OB and have profound reductions in the number of OB neurons and neuronal progenitors (see Fig. S4 in the References supplementary material). Similar results have been obtained Abu-Issa, R., Smyth, G., Smoak, I., Yamamura, K. I. and Meyers, E. N. (2002). Fgf8 is required for pharyngeal arch and cardiovascular development from studies of other mutant mouse strains in which OBs fail in the mouse. Development 129, 4613-4625. to form during development because of defects in genes other Bachler, M. and Neubuser, A. (2001). Expression of members of the Fgf than Fgf8 [e.g. extra-toesJ (XtJ) mutant mice (Sullivan et al., family and their receptors during midfacial development. Mech. Dev. 100, 1995)]. These observations indicate that Fgf8 regulates 313-316. neurogenesis in the OE independently from its regulation of Beites, C. L., Kawauchi, S., Crocker, C. E. and Calof, A. L. (2005). Identification and molecular regulation of neural stem cells in the olfactory neurogenesis in the OB. Taken together, the data support the epithelium. Exp. Cell Res. 306, 309-316. conclusion that defects in OE neurogenesis observed in Fgf8 Calof, A. L., Bonnin, A., Crocker, C., Kawauchi, S., Murray, R. C., Shou, mutants in the present study are due to direct effects of loss of J. and Wu, H. H. (2002). Progenitor cells of the olfactory receptor neuron endogenous Fgf8 on developing OE, and not to indirect effects lineage. Microsc. Res. Tech. 58, 176-188. Cau, E., Gradwohl, G., Fode, C. and Guillemot, F. (1997). Mash1 activates resulting from failure of OB formation. a cascade of bHLH regulators in olfactory neuron progenitors. Development 124, 1611-1621. What cell type is dying in Fgf8 mutants? Chi, C. L., Martinez, S., Wurst, W. and Martin, G. R. (2003). The isthmic Our data from adjacent serial sections indicate that it is Fgf8- organizer signal FGF8 is required for cell survival in the prospective expressing cells themselves that undergo apoptosis in the midbrain and cerebellum. Development 130, 2633-2644. Crossley, P. H. and Martin, G. R. (1995). The mouse Fgf8 gene encodes a absence of FGF8 signaling (Fig. 6). The results of in situ family of polypeptides and is expressed in regions that direct outgrowth and hybridization analysis using probes to intronic versus exonic patterning in the developing embryo. Development 121, 439-451. sequences indicate that ectodermal cells that initially transcribe Cuschieri, A. and Bannister, L. H. (1975). The development of the olfactory Fgf8 RNA give rise to a larger population of neuroepithelial mucosa in the mouse: light microscopy. J. Anat. 119, 277-286. DeHamer, M. K., Guevara, J. L., Hannon, K., Olwin, B. B. and Calof, A. cells, some of which continue to express processed Fgf8 L. (1994). Genesis of olfactory receptor neurons in vitro: regulation of mRNA (Fig. 1C). Taken together with double-label in situ progenitor cell divisions by fibroblast growth factors. Neuron 13, 1083- hybridization results showing overlap between the domains of 1097. 5222 Development 132 (23) Research article

Dickinson, R. J., Eblaghie, M. C., Keyse, S. M. and Morriss-Kay, G. M. Mesenchymal/epithelial induction mediates olfactory pathway formation. (2002). Expression of the ERK-specific MAP kinase phosphatase Neuron 28, 411-425. PYST1/MKP3 in mouse embryos during morphogenesis and early Lee, S. M., Danielian, P. S., Fritzsch, B. and McMahon, A. P. (1997). organogenesis. Mech. Dev. 113, 193-196. Evidence that FGF8 signalling from the midbrain-hindbrain junction Dono, R., Texido, G., Dussel, R., Ehmke, H. and Zeller, R. (1998). Impaired regulates growth and polarity in the developing midbrain. Development 124, cerebral cortex development and blood pressure regulation in FGF-2- 959-969. deficient mice. EMBO J. 17, 4213-4225. Mahmood, R., Bresnick, J., Hornbruch, A., Mahony, C., Morton, N., Downward, J. (1998). Ras signalling and apoptosis. Curr. Opin. Genet. Dev. Colquhoun, K., Martin, P., Lumsden, A., Dickson, C. and Mason, I. 8, 49-54. (1995). A role for FGF-8 in the initiation and maintenance of vertebrate limb Drossopoulou, G., Lewis, K. E., Sanz-Ezquerro, J. J., Nikbakht, N., bud outgrowth. Curr. Biol. 5, 797-806. McMahon, A. P., Hofmann, C. and Tickle, C. (2000). A model for Marigo, V. and Tabin, C. J. (1996). Regulation of patched by sonic hedgehog anteroposterior patterning of the vertebrate limb based on sequential long- in the developing neural tube. Proc. Natl. Acad. Sci. USA 93, 9346-9351. and short-range Shh signalling and Bmp signalling. Development 127, 1337- Maruoka, Y., Ohbayashi, N., Hoshikawa, M., Itoh, N., Hogan, B. M. and 1348. Furuta, Y. (1998). Comparison of the expression of three highly related Dubrulle, J. and Pourquie, O. (2004). Fgf8 mRNA decay establishes a genes, Fgf8, Fgf17 and Fgf18, in the mouse embryo. Mech. Dev. 74, 175- gradient that couples axial elongation to patterning in the vertebrate embryo. 177. Nature 427, 419-422. Mathis, L., Kulesa, P. M. and Fraser, S. E. (2001). FGF receptor signalling Eblaghie, M. C., Lunn, J. S., Dickinson, R. J., Munsterberg, A. E., Sanz- is required to maintain neural progenitors during Hensen’s node progression. Ezquerro, J. J., Farrell, E. R., Mathers, J., Keyse, S. M., Storey, K. and Nat. Cell Biol. 3, 559-566. Tickle, C. (2003). Negative feedback regulation of FGF signaling levels by Meyers, E. N., Lewandoski, M. and Martin, G. R. (1998). An Fgf8 mutant Pyst1/MKP3 in chick embryos. Curr. Biol. 13, 1009-1018. allelic series generated by Cre- and Flp-mediated recombination. Nat. Eckenstein, F. P. (1994). Fibroblast growth factors in the nervous system. J. Genet. 18, 136-141. Neurobiol. 25, 1467-1480. Miho, Y., Kouroku, Y., Fujita, E., Mukasa, T., Urase, K., Kasahara, T., Ellis, P., Fagan, B. M., Magness, S. T., Hutton, S., Taranova, O., Hayashi, Isoai, A., Momoi, M. Y. and Momoi, T. (1999). bFGF inhibits the S., McMahon, A., Rao, M. and Pevny, L. (2004). SOX2, a persistent activation of caspase-3 and apoptosis of P19 embryonal carcinoma cells marker for multipotential neural stem cells derived from embryonic stem during neuronal differentiation. Cell Death Differ. 6, 463-470. cells, the embryo or the adult. Dev. Neurosci. 26, 148-165. Murray, R. C. and Calof, A. L. (1999). Neuronal regeneration: lessons from Farbman, A. I. (1992). Cell Biology of Olfaction. Cambridge, UK: Cambridge the olfactory system. Semin. Cell Dev. Biol. 10, 421-431. University Press. Murray, R. C., Navi, D., Fesenko, J., Lander, A. D. and Calof, A. L. (2003). Ford-Perriss, M., Abud, H. and Murphy, M. (2001). Fibroblast growth Widespread defects in the primary olfactory pathway caused by loss of factors in the developing central nervous system. Clin. Exp. Pharmacol. mash1 function. J. Neurosci. 23, 1769-1780. Physiol. 28, 493-503. Niehrs, C. and Meinhardt, H. (2002). Modular feedback. Nature 417, 35-36. Frank, D. U., Fotheringham, L. K., Brewer, J. A., Muglia, L. J., Tristani- Niswander, L., Jeffrey, S., Martin, G. R. and Tickle, C. (1994). A positive Firouzi, M., Capecchi, M. R. and Moon, A. M. (2002). An Fgf8 mouse feedback loop coordinates growth and patterning in the vertebrate limb. mutant phenocopies human 22q11 deletion syndrome. Development 129, Nature 371, 609-612. 4591-4603. Ohkubo, Y., Chiang, C. and Rubenstein, J. L. (2002). Coordinate regulation Fukuchi-Shimogori, T. and Grove, E. A. (2001). Neocortex patterning by and synergistic actions of BMP4, SHH and FGF8 in the rostral the secreted signaling molecule FGF8. Science 294, 1071-1074. prosencephalon regulate morphogenesis of the telencephalic and optic Fukuchi-Shimogori, T. and Grove, E. A. (2003). Emx2 patterns the vesicles. Neuroscience 111, 1-17. Development neocortex by regulating FGF positional signaling. Nat. Neurosci. 6, 825- Ornitz, D. M. (2000). FGFs, heparan sulfate and FGFRs: complex interactions 831. essential for development. BioEssays 22, 108-112. Galli, L. M., Willert, K., Nusse, R., Yablonka-Reuveni, Z., Nohno, T., Ornitz, D. M. and Itoh, N. (2001). Fibroblast growth factors. Genome Biol. Denetclaw, W. and Burrus, L. W. (2004). A proliferative role for Wnt-3a 2, 3005.1-3005.12 in chick somites. Dev. Biol. 269, 489-504. Ortega, S., Ittmann, M., Tsang, S. H., Ehrlich, M. and Basilico, C. (1998). Garel, S., Huffman, K. J. and Rubenstein, J. L. (2003). Molecular Neuronal defects and delayed wound healing in mice lacking fibroblast regionalization of the neocortex is disrupted in Fgf8 hypomorphic mutants. growth factor 2. Proc. Natl. Acad. Sci. USA 95, 5672-5677. Development 130, 1903-1914. Panman, L. and Zeller, R. (2003). Patterning the limb before and after SHH Graham, V., Khudyakov, J., Ellis, P. and Pevny, L. (2003). SOX2 functions signalling. J. Anat. 202, 3-12. to maintain neural progenitor identity. Neuron 39, 749-765. Platt, K. A., Michaud, J. and Joyner, A. L. (1997). Expression of the mouse Grindley, J. C., Davidson, D. R. and Hill, R. E. (1995). The role of Pax-6 Gli and Ptc genes is adjacent to embryonic sources of hedgehog signals in eye and nasal development. Development 121, 1433-1442. suggesting a conservation of pathways between flies and mice. Mech. Dev. Grove, E. A. and Fukuchi-Shimogori, T. (2003). Generating the cerebral 62, 121-135. cortical area map. Annu. Rev. Neurosci. 26, 355-380. Reuss, B. and von Bohlen und Halbach, O. (2003). Fibroblast growth factors Hebert, J. M. and McConnell, S. K. (2000). Targeting of cre to the Foxg1 and their receptors in the central nervous system. Cell Tissue Res. 313, 139- (BF-1) locus mediates loxP recombination in the telencephalon and other 157. developing head structures. Dev. Biol. 222, 296-306. Shou, J., Murray, R. C., Rim, P. C. and Calof, A. L. (2000). Opposing effects Hsu, P., Yu, F., Feron, F., Pickles, J. O., Sneesby, K. and Mackay-Sim, A. of bone morphogenetic proteins on neuron production and survival in the (2001). Basic fibroblast growth factor and fibroblast growth factor receptors olfactory receptor neuron lineage. Development 127, 5403-5413. in adult olfactory epithelium. Brain Res. 896, 188-197. Smart, I. H. (1971). Location and orientation of mitotic figures in the Irving, C. and Mason, I. (2000). Signalling by FGF8 from the isthmus developing mouse olfactory epithelium. J. Anat. 109, 243-251. patterns anterior hindbrain and establishes the anterior limit of Hox gene Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre expression. Development 127, 177-186. reporter strain. Nat. Genet. 21, 70-71. Kawauchi, S., Takahashi, S., Nakajima, O., Ogino, H., Morita, M., Storm, E. E., Rubenstein, J. L. and Martin, G. R. (2003). Dosage of Fgf8 Nishizawa, M., Yasuda, K. and Yamamoto, M. (1999). Regulation of lens determines whether cell survival is positively or negatively regulated in the fiber cell differentiation by transcription factor c-Maf. J. Biol. Chem. 274, developing forebrain. Proc. Natl. Acad. Sci. USA 100, 1757-1762. 19254-19260. Sullivan, S. L., Bohm, S., Ressler, K. J., Horowitz, L. F. and Buck, L. B. Kawauchi, S., Beites, C., Crocker, C. E., Wu, H. H., Bonnin, A., Murray, (1995). Target-independent pattern specification in the olfactory epithelium. R. C. and Calof, A. L. (2004). Molecular signals regulating proliferation Neuron 15, 779-789. of stem and progenitor cells in mouse olfactory epithelium. Dev. Neurosci. Sun, X., Meyers, E. N., Lewandoski, M. and Martin, G. R. (1999). Targeted 26, 166-180. disruption of Fgf8 causes failure of cell migration in the gastrulating mouse Key, B., Treloar, H. B., Wangerek, L., Ford, M. D. and Nurcombe, V. embryo. Genes Dev. 13, 1834-1846. (1996). Expression and localization of FGF-1 in the developing rat olfactory Sun, X., Mariani, F. V. and Martin, G. R. (2002). Functions of FGF system. J. Comp. Neurol. 366, 197-206. signalling from the apical ectodermal ridge in limb development. Nature LaMantia, A. S., Bhasin, N., Rhodes, K. and Heemskerk, J. (2000). 418, 501-508. Fgf8 is required for olfactory neurogenesis 5223

Tanaka, A., Miyamoto, K., Minamino, N., Takeda, M., Sato, B., Matsuo, H. and Matsumoto, K. (1992). Cloning and characterization of an androgen-induced growth factor essential for the androgen-dependent growth of mouse mammary carcinoma cells. Proc. Natl. Acad. Sci. USA 89, 8928-8932. Temple, S. and Qian, X. (1995). bFGF, neurotrophins, and the control or cortical neurogenesis. Neuron 15, 249-252. Trainor, P. A., Ariza-McNaughton, L. and Krumlauf, R. (2002). Role of the isthmus and FGFs in resolving the paradox of neural crest plasticity and prepatterning. Science 295, 1288-1291. Trumpp, A., Depew, M. J., Rubenstein, J. L., Bishop, J. M. and Martin, G. R. (1999). Cre-mediated gene inactivation demonstrates that FGF8 is required for cell survival and patterning of the first branchial arch. Genes Dev. 13, 3136-3148. Wood, H. B. and Episkopou, V. (1999). Comparative expression of the mouse Sox1, Sox2 and Sox3 genes from pre-gastrulation to early somite stages. Mech. Dev. 86, 197-201. Wu, H. H., Ivkovic, S., Murray, R. C., Jaramillo, S., Lyons, K. M., Johnson, J. E. and Calof, A. L. (2003). Autoregulation of neurogenesis by GDF11. Neuron 37, 197-207. Xu, J., Liu, Z. and Ornitz, D. M. (2000). Temporal and spatial gradients of Fgf8 and Fgf17 regulate proliferation and differentiation of midline cerebellar structures. Development 127, 1833-1843. Zuniga, A., Haramis, A. P., McMahon, A. P. and Zeller, R. (1999). Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds. Nature 401, 598-602. Development